Anti-sickling agents

ABSTRACT

Compounds for the treatment of sickle-cell disease, and methods for their use are provided. The compounds have a dual mode of action. First, binding of the compounds to hemoglobin increases the oxygen affinity of both normal and sickle hemoglobin. Secondly, binding of these compounds to the N-terminal amino acid of sickle hemoglobin results in destabilization of potential contacts between sickle hemoglobin molecules, preventing polymerization and the formation of fibrous precipitates of the sickle hemoglobin. The compounds are also useful for inducing hypoxia, e.g. to augment cancer treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of currently pending U.S.patent application Ser. No. 10/725,935, filed Dec. 3, 2003, the completecontents of which are hereby incorporated by reference.

This invention has been made in part by funds from government sources,including grant numbers K01HL04367 and R01HL65715 from the NationalInstitutes of Health. The United States government may have certainrights to this invention.

DESCRIPTION BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to agents for the treatment ofsickle-cell disease. In particular, the invention provides 5-memberedheterocyclic anti-sickling agents that are highly effective andnon-toxic, and methods for their use.

2. Background of the Invention

Sickle cell disease is one of the most prevalent hematologic geneticdisorders in the world (Ingram, 1956; Pauling, et al. 1949) that occursas a result of a single point mutation of Glu6 in Hb to Val6 in sicklehemoglobin (HbS). Two quaternary structures are known for Hb, the deoxyconformation (tense), and the oxygenated conformation (relaxed). Whenthe allosteric equilibrium is shifted toward the relaxed state, ahigh-affinity Hb is obtained that readily binds and holds oxygen, whilethe converse is true for the tense state. Perutz (1970) and Baldwin &Chothia (1979) elucidated at atomic resolution the tetrameric structuresof the tense (T) and relaxed (R) forms of Hb. The tetramer is composedof two αβ dimers that are arranged around a twofold axis of symmetry.This arrangement yields a central water cavity, with two openings; theα- and β-clefts. The source of the tension in the T state is due tocrosslinking salt bridges and hydrogen bonds between the subunits, aswell as preferential binding of an indigenous allosteric effector of Hb,2,3-diphosphoglycerate (2,3-DPG) that stabilize the T state by formingsalt bridges between the two β-subunits (Arnone, 1992). The T-Rtransition occurs as a result of uptake of oxygen which leads to thedisruption of many of the T state intersubunit interactions, as well asexpulsion of the 2,3-DPG. The allosteric transition results in arotation of the α1β1 dimer relative to the α2β2 dimer by 12-15° (Baldwin& Chothia, 1979). The R state structure has a smaller central watercavity, as well as fewer intersubunit salt bridges and hydrogen bonds.For a long period of time, the allosteric equilibrium of Hb embodied inthe two-state MWC model (Monod, et. al., 1965) was believed to involveonly the T-R transition, and the R state quaternary structure wasthought to be the only relaxed conformer. However, recentcrystallographic and other studies have revealed the existence of multirelaxed Hb states, including R2 and others that exist in solution with R(Silva, et al. 1992; Smith, et al., 1991; Mueser, et al., 2000). Thereis still a controversy about the physiological importance of all theserelaxed states, and how they relate to one another in Hb allostery.Silva et al., (1992) and Smith et al., (1991) suggested that the R2quaternary structure is an intermediate between the T and R structures.Further analysis has shown that R2 is not an intermediate in the T to Rtransition, but rather, it is another relaxed end-state structure (Janin& Wodak, 1993; Doyle, et al., 1992). Srinivasan & Rose (1994) havefurther suggested that R2 may be the physiologically relevant end stateand that the R structure is an intermediate structure trapped betweenthe R2 and T states by the high-salt crystallization conditions. Incontrast, the R2 structure formation is believed to be favored bylow-salt that mimic the in vivo condition (Silva et al., 1992;Srinivasan & Rose, 1994).

Hb and HbS have almost identical positions for all amino acids, even inthe A helix of the chains where the mutation occurs. The presence of theVal6 results in hydrophobic interaction between the mutation region ofone Hb molecule and a region defined by Phe85 and Leu88 in the hemepocket of another Hb molecule. This interaction occurs only in thedeoxygenated HbS (deoxyHbS), and induces polymerization of the deoxyHbSmolecules into fibers. The formation of HbS polymers causes the normallyflexible red blood cells to adopt rigid, sickle like shapes that blocksmall capillaries and cause both local tissue damage and severe pain.The disease is also characterized by other symptoms, includinghemolysis, which gives rise to anemia and jaundice, elevation ofbilirubin level leading to high incidence of gall stones and impairmentof hepatic excretory function. Other clinical features include legulceration, pneumonia, enlarged liver and spleen. Other studies on thegellation of deoxyHbS and various Hb variants have also provided crucialinformation on other contact points on the Hb that are important instabilizing the HbS fibre (Adachi & Asakura, 1980; Bunn, et al., 1986).There are various therapeutic strategies to treat sickle cell disease(SCD), including (1) Pharmacological modulation of fetal hemoglobin(HbF): HbF has been shown to decrease HbS polymerization, and there areseveral agents that are known to induce HbF formation by possiblyreactivating the genetic switch for HbF (Olivieri & Weatherall, 1998).Examples of such agents include 5-azacytidine, hydroxyurea and cytosinearabinoside (Mehanna, 2001). Unfortunately, there are serious toxic sideeffects associated with this therapy as a result of high doses andfrequency of administration (Edelstein, 1985), (2) Bone marrowtransplantation: Bone marrow transplant has also been used as a totalgene replacement therapy for HbS in extreme cases (Hillery, 1998,Johnson, 1985). This approach is very expensive and has its own inherenttoxicities and risks (Hillery, 1998), (3) Blood transfusion: This is oneof the most common SCD therapies, however, repeated blood transfusionsare known to be associated with the risks of infectious diseases, ironoverload and allergic reactions (Ballas, 1999), (4) Opioid analgesics:This therapy is necessary to deal with pain crisis, however, opioidtherapy often results in addiction and/or seizures and/or depression,(Ballas, 1999), (5) Erythrocyte membrane acting agents: Since thesickling process is partly dependent on intracellular concentration ofsickle Hb, agents that induce cell swelling (Asakura, 1980) or inhibitcell dehydration (Orringer & Berkwitz, 1986) could decrease the HbSconcentration and help delay the polymerization process, and (6)Antigelling agent or HbS modifiers: These compounds interfere with themechanism of polymerization by either binding directly to or nearcontact site(s) of the deoxyHbS to inhibit the polymerization process oract directly on HbS to shift the allosteric equilibrium to the moresoluble high-affinity HbS.

In blood, Hb is in equilibrium between the T and the relaxed states. TheFfb delivers oxygen via an allosteric mechanism, and the ability for theHb to release or take oxygen can be regulated by allosteric effectors.The allosteric equilibrium between the T and relaxed states (FIG. 1)shows a typical oxygen equilibrium curve (OEC) for Hb, i.e. a plot ofthe percentage of oxygen bound by Hb against the partial pressure ofoxygen. When the allosteric equilibrium is shifted towards the relaxedstate (left shift of the curve), a high-affinity Hb is obtained thatmore readily binds and holds oxygen while a shift toward the T state(right shift of the curve) results in a low-affinity Hb that more easilyreleases oxygen. An increase in the naturally occurring allostericeffector, 2,3-DPG in red cells right shifts the OEC as does an increasein temperature and decrease in pH (Reeves, 1980). An increase in pH andlowering of the temperature and DPG levels left shifts the OEC. Thedegree of shift in the OEC is reported as an increase or decrease in P₅₀(partial pressure of oxygen at 50% Hb saturation). Regulating theallosteric equilibrium to the relaxed conformation has been of been ofinterest in medicine. In particular, the identification of non-toxiccompounds that efficiently bind to HbS and produce high-affinity HbSwhich does not polymerize have been clinically evaluated as antisicklingagents to treat SCD. There is an ongoing need to identify such compoundsto be used as antisickling agents to treat sickle cell anemia. See, forexample, the use of vanillin (Abraham, 1991), 12C79 (Fitzharris, 1985),furfural (Zaugg, et al., 1997), and substituted isothiocyanates (Park,et al. 2003).

SUMMARY OF THE INVENTION

The present invention provides compounds that are highly effective,specific and non-toxic anti-sickling agents, as well as prodrug forms ofthe compounds. The compounds are based on naturally occurring5-hydroxymethyl-2-furfuraldehyde (5HMF or AMS-13),5-Ethyl-2-furfuraldehyde (5EF), 5-Methyl-2-furfuraldehyde (5MF) and2-furfuraldehyde (FUF), and include analogues and derivatives of thesecompounds. Methods for using the compounds to treat sickle cell diseaseare also provided.

In addition, the invention provides 5-membered heterocyclic aldehydicand protected aldehydic compounds that covalently bind to and modifyhemoglobin. The compounds interact with the amino-terminal amino acid ofnormal and sickle hemoglobins, increasing the oxygen affinity of thehemoglobin. In the case of sickle hemoglobin, binding of the compoundsincreases the fraction of soluble high-affinity oxygenated sicklehemoglobin and/or destabilizes contact between the sickle hemoglobinmolecules, preventing them from sticking together. As a result, thesickle cell disease symptoms are prevented. Thus, methods for using thecompounds to treat sickle cell disease are also contemplated. Further,these compounds may be used to induce hypoxia in tissues, a propertythat is useful in conjunction with certain cancer therapies (e.g. theuse of bioreductive agents) and to help prevent damage caused byradiation therapy.

The invention provides compound of formula

-   -   where R1 is CHO or an aldehyde protecting group; R2 and R3 are        the same or different and are H, OH, alkyl, alkoxy,        hydroxy-alkyl, halogen, aryl, or O-aryl; R4 and R5 are the same        or different and are a substituted or unsubstituted aromatic or        heteroaromatic moiety, a substituted or unsubstituted alkyl or        heteroalkyl ring moiety, a substituted or unsubstituted alkyl or        alkylnoic acid or ester moiety; m=1-6; X=NH, O, S, Se, or P; and        wherein Y =a chemical bridge which includes one to four chemical        moieties selected from the group consisting of CH₂, CO, O, S,        NH, NHCO and NHCONH; Z=(CH)n where n=1-4; and positions of Y and        Z are interchangeable. Exemplary embodiments of the compound are        as follows:        In a preferred embodiment of the invention, R1 is a heterocyclic        ring aldehyde protecting group, and the compound is of the        formula        examples of which include        In other preferred embodiments, R1 is a heterocyclic ring        aldehyde protecting group, and the compound is of the formula        wherein R6 and R7=H or alkyl or ester and may be the same or        different, examples of this embodiment being

The invention also provides a method for treating sickle cell disease ina patient in need thereof, comprising the step of administering to saidpatient a compound of formula

-   -   where R1 is CHO or an aldehyde protecting group; R2 and R3 are        the same or different and are H, OH, alkyl, alkoxy,        hydroxy-alkyl, halogen, aryl, or O-aryl; R4 and R5 are the same        or different and are a substituted or unsubstituted aromatic or        heteroaromatic moiety, a substituted or unsubstituted alkyl or        heteroalkyl ring moiety, a substituted or unsubstituted alkyl or        alkylnoic acid or ester moiety; m=1-6; X=NH, O, S, Se, or P; and        wherein Y =a chemical bridge which includes one to four chemical        moieties selected from the group consisting of CH₂, CO, O, S,        NH, NHCO and NHCONH; Z=(CH)n where n=1-4; and positions of Y and        Z are interchangeable. The compound is administered in        sufficient quantity to ameliorate symptoms of sickle cell        disease. In exemplary embodiments of the method, the compound is        In a preferred embodiment of the method, the compound is        examples of which include        In another embodiment of the method, the compound is        exemplary embodiments being

The invention further provides a method for inducing hypoxia in tissue,comprising the step of administering to the tissue a compound of formula

-   -   where R1 is CHO or an aldehyde protecting group; R2 and R3 are        the same or different and are H, OH, alkyl, alkoxy,        hydroxy-alkyl, halogen, aryl, or O-aryl; R4 and R5 are the same        or different and are a substituted or unsubstituted aromatic or        heteroaromatic moiety, a substituted or unsubstituted alkyl or        heteroalkyl ring moiety, a substituted or unsubstituted alkyl or        alkylnoic acid or ester moiety; m=1-6; X=NH, O, S, Se, or P; and        wherein Y=a chemical bridge which includes one to four chemical        moieties selected from the group consisting of CH₂, CO, O, S,        NH, NHCO and NHCONH; Z=(CH)n where n=1-4; and positions of Y and        Z are interchangeable. According to the method, the compound is        administered in a quantity sufficient to induce hypoxia. The        method may be used to potentiate the action of bioreductive        agents or hyperthermia during cancer treatment, or to protect        against radiation damage during X-ray radiation therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The effects of temperature, pH, and DPG levels on the Hb oxygenequilibrium curve.

FIG. 2. Structures of some furanic as well as other compounds discussedin the application.

FIG. 3. Oxygen equilibrium curves of suspensions of SS cells in theabsence (1) and presence of 5 mM vanillin (2), FUF (3) and 5HMF (4).Note the significant left shift caused by addition of 5HMF.

FIG. 4. Cation-exchange HPLC studies of the hemolysate from the5HMF-reacted SS cells. Peak 1 is normal HbS, while peak 3 is themodified HbS. Peak 2 represents HbS1, a minor component that separatesand elutes earlier from the major HbS peak.

FIG. 5. Kaplan-Meier Survival plot of control and 5HMF (AMS-13) -treatedTg sickle mice.

FIG. 6. Mean survival time of control and 5HMF (AMS-13)-treated Tgsickle mice.

FIG. 7. The effect of 5HMF (AMS- 13) on the percentage of sickled cellsin the arterial blood of Tg sickle mice that were exposed to severehypoxia (5% oxygen). Without treatment (Control), the percentage ofsickled cells increased sharply and the animal died within 15 min.Pretreatment of the mice with 5HMF prolonged the survival timesignificantly. The drug also reduced the percentage of sickled cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides antisickling agents. In some embodiments,the agents are 5-membered heterocyclic compounds that are based on thenaturally occurring substances 5HMF, 5EF, 5MF and FUF. The compoundsinclude analogues and derivatives of 5HMF, 5EF, 5MF and FUF. By“analogue” and “derivative” we mean compounds that possess the samebasic structure as the parent molecule, but in which the various Rgroups as depicted in the Formulas below are replaced as indicated inthe descriptions of the formulas.

The compounds of the present invention exhibit high antisicklingactivity, specificity, and very low toxicity. The compounds contain acentral ring moiety and are of general Formula 1

where R1 is CHO, or an aldehyde protecting group that leaves the centralring moiety and allows the central ring moiety to revert to an aldehydein the body (heterocyclic ring moieties being preferred); R2, R3 and R4are the same or different and are H, OH, alkyl, alkoxy, hydroxy-alkyl,halogen, aryl and O-aryl; and X=NH, O, S, Se, and P.

Preferred embodiments of the compounds are given in Formulas 2-7 below,where R1 is shown as CHO, where R2-R4 are shown and are moieties as setforth above, and, with respect to formulas 4 and 5, where R4 is analkoxy or hydroxy-alkyl; R5=H, alkyl, or aryl, and m=1-6.

In preferred embodiments of the invention, the antisickling compoundsare those of Formula 5 where m=1-6 and R5 is hydrogen.

The invention further provides prodrugs of these compounds. By “prodrug”we mean a form of the compound that contains at least one “protective”chemical group, the presence of which protects the aldehyde moiety ofthe compound against metabolism/degradation until the prodrug is in anenvironment appropriate for removal of the protecting group(s) and“release” of the active form of the compound. The overall effect of theprotecting group is to increase the bioavailability of the activealdehydic compound once the compound reverts to the aldehyde. Onepreferred generic prodrug of the compounds of the present invention isrepresented in Formula 8:

Where R2, R3 and R4 are the same or different and can be H, OH, alkyl,alkoxy, hydroxylkyl, halogen, aryl or O-aryl; R6=H or alkyl; R7=H oralkyl; and X=NH, O, S, Se, or P. Use of protecting groups of this type,(e.g. the amino acids cysteine and homocysteine) have the advantage ofresulting in the release of a non-toxic amino acid upon removal of theprotecting group. It should be understood that other aldehyde protectinggroups may be used in the practice of this invention. Protection groupsof this type are taught, for example, in U.S. Pat. No. 6,251,927 to Laiet al., (Jun. 6, 2001) the complete contents of which are herebyincorporated by reference. These include, but are not limited toconversion of the aldehyde to the corresponding imine, alcohol, acetal,ester, macrcocyclic ester/acetal, macrocyclic ester/imine, hemiacetal,and the like.

Preferred variations of this type of compound are given in Formulas9-13:

where R8=H, alkyl or aryl and m=1-6.

A second preferred generic prodrug of the compounds of the presentinvention is shown in Formula 14

where R2, R3 and R4 are the same or different and can be H, OH, alkyl,alkoxy, hydroxylkyl, halogen, aryl or O-aryl; X=NH, O, S, Se, or P; andn=0-4. These prodrugs have the advantage of being made in a relativelyfacile manner, but do not have the advantage of producing a naturalproduct upon release from the prodrug. Variations of this type ofprodrug are given in Formulas 15-19,

where R9=H, alkyl, or aryl and m=1-6.

The present invention also provides a method for treating sickle celldisease, and to treat symptoms of sickle cell disease such as jaundiceand elevated levels of bilirubin. The method involves administering to apatient with sickle cell disease a quantity of at least one compound ofthe present invention sufficient to ameliorate or reduce symptoms of thedisease, e.g. to decrease or eliminate jaundice and elevated bilirubin.The compounds may be administered as the compounds themselves, or in theform of a prodrug.

The compounds of the present invention are potent antisickling agents.Those of skill in the art will recognize that the amount of such anagent that is to be given to a patient will vary depending on severalfactors, including but not limited to blood volume, hematocrit, patientage, gender, weight, overall physical health, presence of other diseaseconditions, the particular compound being administered, and the like.However, the amount will generally be in the range of from about 50 toabout 1000 mg/kg and more preferably in the range of from about 100 toabout 750 mg/kg. For example, an adult sickle cell patient with a bloodvolume of 4 L and 25% hematocrit will need about 378-630 mg/kg of themost potent compound, 5HMF, and more preferably the dose will be in therange from 200-630 mg/kg.

In another embodiment of the invention, the compounds of the inventionhave the generic formula

In compounds represented by Formula 20, R1 is CHO or an aldehydeprotecting group; R2 and R3 are the same or different and are H, OH,alkyl, alkoxy, hydroxy-alkyl, halogen, aryl, or O-aryl; R4 and R5 arethe same or different and are a substituted or unsubstituted aromatic orheteroaromatic moiety, a substituted or unsubstituted alkyl orheteroalkyl ring moiety, a substituted or unsubstituted alkyl oralkylnoic acid or ester moiety; m=1-6; and X=NH, O, S, Se, or P. Furtherin these compounds, Y is a chemical bridge which includes one to fourchemical moieties which may be CH₂, CO, O, S, NH, NHCO or NHCONH; Z is(CH)n where n=1-4; and Y and Z are interchangeable. By “interchangeable”we mean that the position of Y and Z with respect to R4 and R5 may beeither Y-Z (as depicted in Formula 20 above) or Z-Y (not shown), thelatter of which results in a chain with generic formula —O—R4-Z-Y—R5.

Compounds of this type have been designed to interact with theN-terminal amino acid of normal and sickle hemoglobins, and as a result,the compounds have a dual mode of action. First, binding of thecompounds to hemoglobin increases the oxygen affinity of both normal andsickle hemoglobin. Secondly, binding of these compounds to theN-terminal amino acid of sickle hemoglobin results in destabilization ofpotential contacts between sickle hemoglobin molecules, and preventspolymerization and the formation of fibrous precipitates of the sicklehemoglobin. Binding of the compounds thus helps to maintain solubilityof the hemoglobin, and to alleviate symptoms of sickle cell disease thatare brought on by hemoglobin polymerization. The combined effect ofincreasing oxygen affinity and preventing aggregation increases thepotency of the compounds beyond that of other agents that exhibit onlyone or the other of these activities. Therefore, lower doses of thesecompounds may be required to obtain a therapeutic benefit, compared toother chemical therapies. For example, a typical dose of 5HMF is in therange of about 50-about 1000 mg/kg. In contrast, compounds of formula20, which are derivatives of 5HMF, but have a dual mode of action, willrequire doses in the range of about 25-about 500 mg/kg, i.e. roughlyhalf that of the single mode of action agent 5HMF.

The compounds of Formula 20 are thus useful for the treatment of sicklecell diseases, and the present invention provides methods for suchtreatments.

In addition, because of the effect of the 5-membered heterocyclicaldehydic and protected aldehydic compounds on hemoglobin (i.e. shiftingof the allosteric equilibrium in favor of hemoglobin with ahigh-affinity for oxygen) these compounds are useful for inducing tissuehypoxia for any of a variety of beneficial uses. For example, anincrease in tissue hypoxia can potentiate the action of bioreductiveagents that are used in the treatment of cancer, examples of whichinclude: Mitomycin C, EO9, Tirapazamine, RSU1069. The activities ofthese anticancer drugs are very dependent on the tumor environment andare strongly influenced by factors such as the level of activatingenzymes, the level of oxygen and the pH in tumor cells. In fact,bioreductive agents must undergo reduction to form an active cytotoxicspecies. Therefore induction of hypoxia within solid tumors offers thepossibility of increasing the effectiveness of these antitumor agents.Normally, the aldehydes are administered before administration of thebioreductives.

In addition, the ability of the compounds to induce tissue hypoxia isuseful in cancer (and other) treatments that utilize hyperthermia.Hyperthermia is heat treatment in which the temperature of the tissue iselevated artificially with the aim of receiving therapeutic benefits.Hyperthermia is considered to be an adjunct to other treatments.According to the present invention, the prior induction of hypoxia withthe compounds of the invention in, for example, a cancerous tumor, wouldcause the tumor to be more susceptible to the deleterious effects ofhigh temperature. The tumor cells would thus die more rapidly, orpossibly at lower temperatures than would otherwise be required. Thesynergistic effects of hyperthermia combined with chemotherapy and/orradiation have been observed during treatment with bleomycin, cisplatin,cyclophosphamide, melphalan, mitoxantrone, mitomycin C, thiotepa,misonidazole, and 5-thi-D-glucose. Since some of these compounds workbest under hypoxic conditions, combination therapy with the compounds ofthe present invention will be desirable.

Other uses of the compounds of Formula 20 include ameliorating negativeeffects of X-ray radiation. The use of radiation (for example, in thetreatment of cancerous tumors) typically leads to production of oxygenradicals, which can have a harmful effect on the body. Since the bindingof the compounds to hemoglobin increases its affinity for oxygen, aconcomitant reduction of oxygen available to tissues occurs, the oxygenbeing scavenged and retained by the hemoglobin. This leads to a decreasein the formation of oxygen radicals, and thus to a decrease in damagecaused by such radicals.

Several preferred embodiment of this formula are as follows:

In some embodiments, R1 is a heterocyclic ring aldehyde protectinggroup, and the compound is of the formula

Preferred embodiments of Formula 26 include:

In other embodiments of the invention, R1 is a heterocyclic ringaldehyde protecting group, and the compound is of the formula

In this embodiment R6 and R7 are H or alkyl or ester and may be the sameor different. Exemplary compounds of this embodiment include:

The compounds of the present invention may be administered in any formthat is suitable for delivering an active amount of the compound to thepatient being treated. For examples, the compound may be administered assolid pills or capsules, liquids for oral administration, injectableformulations, etc. The compounds may be provided alone or in combinationwith other constituents, and may be provide in pure or salt form (e.g.,organic or inorganic salts, etc.). In addition, the compounds may beformulated with carriers such as aqueous or oil based vehicles, and canbe accompanied by preservatives (e.g., methyl paraben or benzyl alkoniumchloride (BAK)), surfactants (e.g. oleic acid), solvents, elixirs,flavoring agents (in the case of oral delivery), starch, and othermaterials (preferably those which are generally regarded as safe(GRAS)). In a preferred embodiment, the compound will be administered asa preparation for oral administration in an alcohol-based oraqueous-based carrier.

Likewise, the method of administration may be any of a wide variety ofmethods that are well known to those of skill in the art, such asintravenous, intradermal injection, subcutaneous injection,intramuscular injection, intraperitoneal injection; oral, rectal andbuccal delivery; transdermal delivery; inhalation; etc. In a preferredembodiment, the method of administration is oral delivery. Further, thecompounds of the present invention may be administered in conjunctionwith other known sickle cell disease treatments, or treatments for otherrelated or unrelated disease conditions (e.g. treatment for anemia), andwith other beneficial ancillary regimens such as dietary supplements,exercise regimens, and the like.

While a primary use of the anti-sickling agents of the present inventionis to treat sickle cell disease, those of skill in the art willrecognize that the agents may be used in other applications for which itis beneficial to cause destabilization of the tense (T) states ofhemoglobin, and the switching of the allosteric equilibrium in favor ofthe high-affinity Hb in the form of the R2-state Hb. This aspect of theinvention is further illustrated in Example 5 below. For example, theinvention also contemplates the use of the compounds in the treatment ofpatients with cancer. One or more of the compounds would be administeredto a patient in a quantity sufficient to change the oxygen carryingcapacity of the blood to a state beneficial to eradicating the cancer.In addition, similar to the methods described in U.S. Pat. No. 5,705,521to Abraham, which is herein incorporated by reference, the compounds ofthis invention can be administered to a patient that is or is going toundergo radiation therapy to assist in the effectiveness of theradiation to destroy the cancer.

The compounds may also be used for research purposes. In particular, thegeneric compounds may serve as parent structures for rational drugdesign of additional derivatives and analogues. Examples include but arenot limited to other forms of the compounds that are more or lessactive, or more or less stable; that have altered solubility properties;or that have moieties that serve to target the compounds to a desiredlocation, e.g. across the cell membrane. Alternatively, the compoundsprovided here may serve as parent structures for the design of otherforms of prodrugs.

Some of the compounds used in the practice of the present invention arenaturally occurring, for example, 5-Hydroxymethyl-2-furfuraldehyde (5HMFor AMS-13), 5-Ethyl-2-furfuraldehyde (5EF), and5-Methyl-2-furfuraldehyde (5MF) (see FIG. 2). 5HMF is present in manyfoods such as sweet potatoes, fruits, honey, milk, beer, tomatoproducts, cigarette smoke, and coffee, where the concentration sometimesexceeds 6 g/kg. Commercially, 5HMF is prepared from the fructose portionof sugar.

Methods for synthesizing the prodrug compounds of the present inventionare also described herein, with exemplary synthesis schemes being givenin Example 4 below. Such organic synthesis methods of protectingaldehydes are well-known to those of skill in the art.

Methods of synthesizing compounds such as those depicted in Formula 20are given in Example 7 below.

EXAMPLES Example 1 Structural Basis for the Potent Antisickling Effectof a Novel Class of 5-Membered Heterocyclic Aldehydic Compounds: InVitro Tests and X-ray Crystallographic Studies of the Furanic Compounds,5HMF, 5MF, 5EF and FUF

Chemical structures of 5HMF, 5MF, 5EF and FUF are shown in FIG. 2.

Regulating the Hb allosteric equilibrium to the relaxed conformation hasbeen of interest in medicine, as compounds that bind to Hb S thatproduce high-affinity HbS, which do not polymerize have been clinicallyevaluated as antisickling agents to treat sickle cell anemia. Two suchcompounds are vanillin (Abraham, et al., 1991) and 12C79 (Beddell, etal., 1984; Fitzharris, et al., 1985; Orringer, et al., 1986). Bothcompounds are aldehydes and form Schiff base adducts with Hb. Clinicalstudies with 12C79 (FIG. 2) showed that intravenous infusion of thiscompound (40 mg/kg) in patients with SCD resulted in the formafion ofcompound-Hb adducts at levels of more than 30% without any adverseeffect (Orringer, et al., 1988). Vanillin (FIG. 2) is a food flavoringcompound, and because it is relatively non-toxic, makes it a veryattractive therapeutic agent for SCD. A low-resolution structure of Tstate Hb complexed with vanillin showed that the compound binds to twodifferent sites: a primary site near His103, Cys104 and Gln131, and asecondary site between His116 and His117 (Abraham, et al., 1991). Basedon the results of the X-ray crystallographic analysis and functionalstudies, Abraham, et al., (1991) suggested that vanillin acts todecrease the polymerization of HbS by shifting the allostericequilibrium toward the high-affinity Hb S molecule in the form of Rstate; as well as stereospecific inhibition of the polymerization of Tstate HbS. Additional studies of several analogs of vanillin by Abraham,et al., (1995) and Boyiri, et al., (1995) showed that these compounds,unlike vanillin, bind to the N-terminal Val I of T state Hb, andsurprisingly effect opposite shifts in the OEC. Agents such as,5-formylsalicylic acid (FSA, FIG. 2), which form Schiff baseinteractions with the N-terminal Vail nitrogen, and provide groups forboth salt bridge and hydrogen bonding with the opposite dimer (acrossthe Hb two-fold axis), shift the OEC toward the right. In contrast,agents such as, 3,5-dimethyl-4-hydroxybenzaldehyde (DMHB, FIG. 2), whichalso bind to the N-terminal Val residue of T state Hb in a similarfashion as FSA without any salt bridge interactions with the oppositedimer, shift the OEC toward the left.

In the current studies, we combined the use of aldehydic covalentmodifiers of Rb with our knowledge of the molecular regulation of theallosteric equilibrium to produce potent antisickling compounds thatshould be clinically safe. Specifically, we examined5-hydoxymethyl-2-furfural (5HMF) and several of its analogs, includingfurfural (FUF), 5-methyl-2-furfural (5MF) and 5-ethyl-2-furfural (5EF)(all shown in FIG. 2) for their antisickling potencies. These compoundswere found to significantly shift the allosteric equilibrium to thehigh-affinity Rb, and also act as potent inhibitors of homozygous sicklered blood (SS) cell sickling. One of the compounds, 5HMF modifies HbS by70% compared to 15% for vanillin. 5HMF also inhibits in vitro SS cellsickling by 90%, four times more than vanillin. Also, in vivoantisickling studies using sickle cell transgenic (tg) mice show 5HMF toprolong the life of the hypoxic mice about four times longer compared tocontrol. 5HMF is found in everyday food, and has LD₅₀ of 2.5-5.0 g/Kg(US EPA, 1992) compared to 1.58 for vanillin. Thus we now have acompound which is safe, more potent than any known aldehydicantisickling agent, able to transverse red blood cell to react andmodify HbS. In addition, 5HMF makes an ideal scaffold upon which tobuild more potent and safe compounds.

X-ray crystallographic studies of Hb complexed with these compoundsindicate that they form Schiff base adducts in a symmetrical fashionwith the N-terminal αVal1 nitrogens of Hb. Remarkably, two co-crystaltypes were isolated during these experiments: one crystal type was foundto be composed of the low-affinity or tense (T) state Hb quaternarystructure in complex with the compounds; the other crystal type wascomposed of high-affinity or relaxed state Hb (with a R2 quaternarystructure) in complex with the compounds. Furthermore, the examinedheterocyclic aldehydes were found to bind strongly to the R2 state, butweakly to the T state. Crystallization of the same compounds withliganded Hb resulted in only relaxed R state crystals, which alsoindicated weak compound binding.

These results suggest that the examined heterocyclic aldehydes preventpolymerization of sickle hemoglobin (HbS) and inhibit the sickling of SScells by stabilizing HbS in the high-affinity R2 state. Comparing thehigh resolution crystal structures of 5HMF and vanillin bound to Hb,shows 5HMF to bind much stronger, an indication that 5HMF may residelonger at the binding site. This explains why 5HMF is many fold potentthan vanillin. Most importantly the stronger binding of 5HMF to Hb maytranslate into an even longer half-life and increased bioavailabilityfor 5HMF compared to vanillin, with a concomitant decrease in the dosageneeded for therapy. The biological, as well as the crystallographicstudies of the furanic compounds reveal for the first time the exactmolecular mechanism for the antisickling effects of covalently modifyingaldehydes that bind to N-terminal αVal1 nitrogens of Hb. These examinedcompounds also represent a new class of potentially therapeutic agentsfor treating sickle cell disease (SCD).

Experimental Procedure

Materials and General Procedures. The following compounds: vanillin,FUF, 5MF, 5EF and 5HMF were purchased from Aldrich Chemical Company.Normal red blood (AA) cells were collected from adult donors. SS cellswere obtained from patients with SCD. Purified human adult Hb in 50 mMBis Tris buffer, pH 6.8, was prepared from discarded human blood aspreviously described (Safo and Abraham, 2003).

Oxygen Equilibrium Studies with Normal Whole Blood. Normal blood samples(hematocrit 40%) in the presence of 5 mM Vanillin, FUF, 5MF, 5EF and5HMF (solubilized in DMSO) were equilibrated at 37° C. for 1 hr. Thesamples were then incubated in IL 237 tonometers (InstrumentationLaboratories, Inc. Lexington, Mass.) for approximately 10 min at 37° C.,and allowed to equilibrate at oxygen tensions 7, 20, and 60 mmHg. Thesamples were aspirated into an IL 1420 Automated Blood Gas Analyzer andan IL 482 or IL 682 Co-oximeter (Instrumentation Laboratories) todetermine the pH, pCO₂, pO₂ and the Hb oxygen saturation values (sO2).The pO₂ and sO₂ values at each oxygen saturation level were thensubjected to a non-linear regression analysis using the programScientist (Micromath, Salt Lake City, Utah) to calculate the P₅₀ andHill coefficient values (n₅₀). P₅₀ is the oxygen pressure in mmHg atwhich Hb is 50% saturated with oxygen. A dose-response study with 5HMFwas performed at final compound concentrations of 1 and 2 mM.

Oxygen Equilibrium Studies with Homozygous Sickle Red Blood Cells. SScells were suspended in PBS to a final hematocrit of 10%. Vanillin, FUF,5HMF (solubilized in DMSO) was added to this suspension at finalconcentrations of 5 mM and incubated at 37° C. for 1 hr. A 40 uL aliquotof this suspension was added to 4 ml Hemox buffer and subjected tooxygen equilibrium analysis (37° C.) using a Hemox-Analyzer (TCSScientific Corp., Southampton, Pa.) (Asakura, 1979).

Transport through Homozygous Sickle Red Blood Cell Membrane and Reactionwith HbS. The compound-treated SS cells obtained in the precedingexperiment (oxygen equilibrium studies with SS cells) were hemolyzed byadding 5 volumes of 5 mM potassium phosphate buffer, pH 7.4 containing0.5 mM EDTA. After centrifugation, the hemolysate was subjected to bothoxygen equilibrium analysis using Hemox-Analyzer and cation-exchangeHPLC analysis using a Hitachi HPLC apparatus (Model D-7000 Series) and aSwift WCX column (Swift™ WCX-PEEK: 50 mm×4.6 mm, Isco, Inc., Lincoln,Nebr.). The column was developed using a linear gradient of phase B from25% to 90% at 410 nm (Mobile Phase A: 40 mM Bis-Tris, 5 mM EDTA, pH 6.5;Phase B: 40 mM Bis-Tris, 5 mM EDTA, 0.2 M sodium chloride, pH 6.5). TheHbS adduct formation (modification of HbS) values are expressed inpercentages, using the following formula:${{Mod}\quad H\quad b\quad{S(\%)}} = {\frac{{{peak}\quad{area}\quad{of}\quad{modified}\quad H\quad b}\quad}{\left( {{{peak}\quad{area}\quad{of}\quad{modified}\quad H\quad b}\quad + \quad{{peak}\quad{area}\quad{of}\quad{unmodified}\quad H\quad b}} \right.} \times 100}$

Antisickling Studies with Homozygous Sickle Red Blood Cells. The effectsof vanillin, FUF and 5HMF on the inhibition of SS cells sickling wereevaluated as previously described (Asakura & Mayberry, 1984). Briefly,SS cells suspended in buffered saline solution, pH 7.4 (hematocrit of10%) were incubated at 37° C. with 4% oxygen in the presence of 5 mMcompound. Aliquots (10 ul) of the suspensions were obtained after 5 hrsand fixed with 2% glutaraldehyde solution without exposure to air.Morphological analysis and percentage of SS cells that were not sickledwere conducted using a computer-assisted image analysis system asdescribed elsewhere (Hijiya, 1991). Dose-response studies of FUF and5HMF at compound concentrations of 1 and 2 mM were also performed.

The Effect of Compounds on Homozygous Sickle Red Blood Cell Size. Tostudy the effect of the compounds on the degree of hydration/dehydrationof SS cells, the compound-treated SS cells obtained in the precedingexperiment (antisickling studies with SS cells) were evaluated with aHemavet Cell Analyzer to determine the mean corpuscular volume (MCV).

Crystallization Experiments. Crystallization experiments to obtain T andR state crystals were conducted with FUF and 5HMF. The experimentsinvolved 4-25 molar excess of the compounds to Hb (tetramer). For the Tstate crystallization experiment, the compounds solubilized in DMSO wereincubated with deoxy Hb (60 mg/mL protein) for at least 1 hour to, formthe Schiff base adduct. Sodium cyanoborohydride, in 4-25 molar excess ofHb, was added to reduce the reversible Schiff base-adduct to thecorresponding alkylamine covalent bond. Subsequent crystallization ofthe compound-deoxy Hb complex solutions in 10 mL test tubes using3.2-3.6 M sulfate/phosphate precipitant (pH 6.5) was performed in aglove box under nitrogen atmosphere as previously described (Safo, etal., 2003; Perutz, 1968) for obtaining high-salt T state crystals.Reduction of the Schiff base-adduct is necessary to observe the boundcompound crystallographically (Boyiri, et al., 1995). Unexpectedly, theexperiment resulted in two different crystals—a rectangular crystal(space group P2₁), which is isomorphous to T state native crystal, and atrigonal crystal (space group P3₂2₁), which was later determined to havea relaxed state conformation in the form of R2 quaternary structure. TheT state crystals grew between 2-10 days, while the R2 state crystalsgrew between 7-30 days.

Another experiment was designed to crystallize the compound-Hb complexesin R state form using carbonmonoxy Hb (COHb), following a previouslydescribed procedure (Safo, et al., 2003; Perutz, 1968). Oxygenated Hbsolution was evacuated for about 10 minutes, and the resulting deoxy Hbsolution was fully saturated with CO to generate COHb. The compoundssolubilized in DMSO were then reacted with the COHb, followed byaddition of sodium cyanoborohydride to reduce the Schiff base-adduct.Crystallization was carried out with a solution of 30-50 mg/mL protein,3.2-3.4 M Na⁺/K⁺ phosphate, pH 6.4, and two drops of toluene in 10 mltest tubes. The experimental procedures were done under aerobiccondition, and resulted in co-crystals (4-30 days) isomorphous to Rstate native crystals (space group P4₁2₁2).

Data Collection, Processing and Structure Refinement. X-ray diffractiondata sets for the R, R2 and T state co-crystals were collected at 1000 Kusing a Molecular Structure Corporation (MSC) X-Stream Cryogenic CoolerSystem (MSC, The Woodlands, Tex.), a R-Axis II image plate detectorequipped with OSMIC mirrors, and a Rigaku RU-200 generator operating at50 kV and 100 mA. Prior to use in diffraction, the crystals were firstwashed in a cryoprotectant solution containing 50 μL mother liquor and10-12 μL glycerol. The data sets were processed with MSC BIOTEX softwareprogram. All structure refinements and omit maps were performed with theCNS program (Brunger, et. al., 1998). Model building and correction werecarried out using the graphic program TOM (Cambillau & Horjales, 1987).

Structure Determinations and Refinements of the R2 State ComplexStructures. The structure of the FUF-Hb complex in the R2 state crystalwas the first to be determined by molecular replacement method (Navaza,1994) using the α1β1-α2β2 R2 state native Hb structure (PDB code 1BBB)as a search model. The translation function using the space group P3₂21gave a solution of a tetramer in the asymmetric unit with a finalcorrelation coefficient of 69.2 and Rfactor of 35.5% for data between8.0-4.0 Å. Prior to using the R2 structure as a search model, we assumedthe crystal to be a T state Hb in another crystal form. However, the useof a T state structure (PDB code 2HHB) as a search model failed to givea clear solution. A similar search with a R state structure (PDB code 1AJ9) also failed to give a clear solution. The molecular replacementmodel was subjected to a rigid body refinement, followed by conjugategradient minimization and simulated annealing. Strong and cleardensities were identified for two FUF molecules bound at the N-terminalαVal1 residues in a symmetry-related fashion. The N-terminal αVal1binding site is located in the central water cavity of Hb close to themouth of the α-cleft. The electron density from the bound compoundoverlapped that of the αVal1 nitrogen, suggesting a covalent interactionbetween FUF and the nitrogen. The electron density map also showedligation of the four heme Fe atoms, and water ligands were fitted to thedensity. Alternate fitting of O₂ ligands produced distorted geometry ofthe Fe—O—O bonds and angle. Several alternate rounds of conjugategradient minimization, simulated annealing, individual B factorrefinements, and the addition of 7 sulfate anions and 297 watermolecules, with manual model corrections, brought the final Rfactor to21.7% and Rfree to 27.4% at 2.25 Å resolution. The crystallographic datafor the structure is summarized in Table 1.

The starting model for the refinement of the 5HMF-Hb complex structurewas the FUF-Hb structure—after deletion of FUF, water molecules andsulfate anions. A round of rigid body, conjugate gradient minimizationand simulated annealing refinements also showed two 5HMF bound at thetwo symmetry-related N-terminal αVal1 nitrogens. In contrast to theFUF-Hb structure, O₂ molecules were ligated to the Fe atoms. Severalalternate rounds of conjugate gradient minimization, simulatedannealing, individual B factor refinements, and the addition of 7sulfate anions and 538 water molecules, with intermittent manual modelcorrections, brought the final Rfactor to 18.3% and Rfree to 22.3% at1.85 Å resolution. The crystallographic data for the 5HMF-Hb structureis summarized in Table 1. The atomic coordinates and structure factorshave been deposited in the RCSB Protein Data Bank with accession codes1QXD and 1QXE for the FUF- and 5HMF-Hb structures, respectively.

Structure Determinations and Refinements of the T state ComplexStructures. The starting model for the refinement of the T state 5HMF-Hbstructure was the isomorphous α1β1-α2β2 T state native structure (PDBcode 2HHB). After rigid body refinement, and subsequent gradientminimization and simulated annealing, the electron density maps for thestructure, unlike those of the R2 state complex structures, revealedonly weak and undefined densities at the N-terminal αVal1 binding sites.Repeated cycles of refinements, addition of water molecules, and modelbuilding did not show improved density at the binding site tosuccessfully model 5HMF. There were no other apparent binding sites. Thefinal Rfactor and Rfree for the 5HMF-Hb structures are 16.3 and 20.7% at1.86 Å resolution. Other statistics for the crystal are reported inTable 1. TABLE 1 Crystal Information, Data Collection and RefinementParameters for the Hb Complex Structures FUF (R2 state) 5HMF (R2 state)5HMF (T state) FUF (R state) Data collection Space Group P3₂21 P3₂21 P2₁P4₁2₁2 Cell Dimensions (Å) 91.40 91.86 62.61 53.46 91.40 91.86 82.4753.46 142.00 143.53 53.46 192.88 99.52 Mol/asymmetric unit 1 tetramer 1tetramer 1 tetramer 1 dimer Resolution (Å) 69.1-2.25 69.6-1.85 82.5-1.8684.0-2.0 No. of measured refl. 124853 286747 108495 105005 Uniquereflections 32647 56802 41917 18357 I/sigma I 7.0 12.5 15.8 13.2 Compl.(%) 97.0 93.1 90.7 92.0 Rmerge (%)^(a) 7.5 6.9 6.5 6.9 RefinementResolution (Å) 69.1-2.25 69.6-1.85 52.7-1.86 51.5-2.0 Sigma cutoff (F)0.0 0.0 0.0 0.0 No. of refl. 32645 56780 41895 18316 Rfactor (%) 21.718.4 16.3 21.3 Rfree (%)^(b) 27.4 22.3 20.7 26.3 Rmsd standard Geom.Bond-lengths (Å) 0.011 0.013 0.015 0.012 Bond-angles (°) 1.87 1.89 1.721.90 Dihedral angles Most favored regions 91.4 92.8 93.6 92.8 Additionalregions 8.6 7.2 6.4 7.2^(a)Rmerge = Σ(<I> − I)/ΣI.^(b)5% of the reflection which were used for the calculation of Rfreewere excluded from the refinement.

The 5HMF-Hb structure, without water and ligands was used as a startingmodel for the refinement of the FUF-Hb structure. Similar to the 5HMF-Hbstructure, refinements of the FUF-Hb structure did not result in anyinterpretable density at the binding pocket. The structure was notrefined to completion, and detailed statistics for the crystal is notreported in the Table 1.

Structure Determinations and Refinements of the R state ComplexStructures. The isomorphous α1β1 dimer R state structure (1LJW) afterdeletion of water molecules and phosphate anions was used as thestarting model to refine the FUF-Hb structure. Similar to the T statecomplexes, repeated refinements of the FUF-Hb structure with modelbuilding showed only weak and uninterpretable density at the N-terminalcVall binding pocket. The final Rfactor and Rfree are 21.3 and 26.3 at2.0 Å resolution, and detailed statistics for the crystal are reportedin Table 1.

The FUF-Hb structure, without water and ligands was used as a startingmodel for the refinement of the 5HMF-Hb structure. Similar to that ofthe FUF crystal, refinements also showed uninterpretable density at thebinding pocket, and the refinement was aborted. No detailed statisticsfor the crystal are reported in the Table 1.

Results

Vanillin has been clinically tested for SCD therapy, and was studiedwith the examined heterocyclic aldehydes, also referred to as furaniccompounds. We have also previously published detailed functional andantisickling properties of vanillin (Abraham, et al., 1991).Additionally, relatively high concentrations of compounds (5-10 mM) wereused to ensure complete reaction with Hb in the current studies. This isnecessary, as the concentration of Hb within RBCs is approximately 5mmol/L, and at 25% hematocrit with a blood volume of 4 L, 10 mmol/Lcompound is needed to produce a 2:1 compound:Hb adduct. There are twoidentical binding sites in Hb since it possesses a two-fold axis ofsymmetry.

Oxygen Equilibrium Studies with Normal Whole Blood. Table 2 summarizesthe effects of four furanic compounds and vanillin (at 5 mMconcentrations) on AA cell. Allosteric effectors that increase Hb oxygenaffinity decrease the P₅₀ (left-shift the OEC)—relative to the control.This results in a negative ΔP₅₀ value. The most potent compound is 5HMF(ΔP₅₀=−17.52 mmHg), followed by 5MF (ΔP₅₀=−16.16 mmHg), 5EF (ΔP₅₀=−15.71mmHg), and FUF (−11.35 mmHg). The poorest left shifting compound isvanillin (−6.78 mmHg). Table 2 also shows that 5HMF left-shifts the OECin a dose-dependent manner. The Hill coefficients of the modified Hbs,with the exception of that of FUF are smaller compared to that of AAcells alone. TABLE 2 Oxygen Equilibrium Studies with Normal WholeBlood^(a) ΔP₅₀ Compound^(b) P₅₀ (mmHg)^(c) (mmHg)^(d) n₅₀ ^(e) Control25.84 ± 0.01 — 2.27 ± 0.20 5 mM Vanillin^(e) 19.06 ± 1.98 −6.78 1.98 ±0.23 5 mM FUF 14.49 ± 0.06 −11.35 2.30 ± 0.01 5 mM 5MF  9.68 ± 0.24−16.16 1.70 ± 0.06 5 mM 5EF 10.13 ± 0.75 −15.71 1.77 ± 0.14 1 mM 5HMF19.19 ± 1.51 −6.65 2.08 ± 0.14 2 mM 5HMF 15.04 ± 0.51 −10.80 1.98 ± 0.085 mM 5HMF  8.32 ± 0.40 −17.52 1.88 ± 0.06^(a)The results are the means ± S.E. for 2 measurements.^(b)The ratio of compound to Hb at 1 mM, 2 mM and 5 mM compoundconcentrations are 0.8, 1.6 and 4, respectively.^(c)P₅₀ is the oxygen pressure at which AA cells (40% hematocrit) in theabsence or presence of compound is 50% saturated with oxygen.^(d)ΔP₅₀ is P₅₀ of compound treated AA cells-P₅₀ of control.^(e)n₅₀ is the Hill coefficient at 50% saturation with oxygen.

Oxygen Equilibrium Studies with Homozygous Sickle Red Blood Cells. Table3 (columns 2 and 3) summarizes changes in P₅₀ and ΔP50 for SS cellstreated with vanillin, FUF, and 5HMF at 5 mM concentration. Allcompounds shift the OEC to the left, and as observed in the oxygenequilibrium studies with AA cells, 5HMF is allosterically the mostpotent compound (ΔP₅₀=−25.2 mmHg) followed by FUF (ΔP₅₀=−15.8 mmHg), andlastly vanillin (ΔP₅₀=−13.5 mmHg). FIG. 3 shows the OEC curve for allthree compounds at 5 mM concentration, and shows that 5HMF issignificantly left-shifted compared with the OEC of both FUF andvanillin. As previously observed for vanillin with both AA and SS cells(Abraham, et al., 1991), higher concentrations of tested compoundsresulted in a more hyperbolic OEC from the normal sigmoidal shapedcurve. TABLE 3 Oxygen Equilibrium and Adduct Formation Studies withHomozygous Sickle Red Blood Cells^(a) SS cells SS cells HemolysateHemolysate HbS adduct Compound P₅₀ (mmHg)^(b) ΔP₅₀ (mmHg)^(c) P₅₀ (mmHg)ΔP₅₀ (mmHg)^(d) (%)^(e) Control 31.2 ± 1.0 — 11.2 ± 0.2  — — Vanillin17.7 ± 2.2 −13.5 8.1 ± 1.1 −3.1 15 ± 3.6 FUF 15.4 ± 1.7 −15.8 5.3 ± 0.6−5.9 24 ± 5.7 5HMF  6.0 ± 1.2 −25.2 1.8 ± 0.3 −9.4  70 ± 10.0^(a)The results are the means ± S.E. for 2 measurements.^(b)P₅₀ is the oxygen pressure at which SS cells (10% hematocrit) orhemolysate (in the absence or presence of compound solubilized in DMSO)is 50% saturated with oxygen.^(c)ΔP₅₀ is P₅₀ of compound treated SS cells or hemolysate-P₅₀ ofcontrol.^(d)P₅₀ values obtained from hemolysate after incubation of compoundswith SS cells.^(e)HbS adduct values obtained from HPLC elution patterns of hemolysateafter incubation of compounds with SS cells.

Transport through Homozygous Sickle Red Blood Cell Membrane and Reactionwith HbS. These experiments were undertaken to determine if theleft-shift observed for compound-treated SS cells is the result of adirect interaction of the compound with HbS, and also to determine ifP₅₀ changes observed in SS cells treated with test compounds isattributed to the formation of different levels of compound-HbS adduct.The results are summarized in Table 3 (columns 4-6). Each of the testedcompounds produces a new HbS modified peak that eluted before that ofthe parent HbS peak, indicating the formation of covalently modified HbSadducts. 5HMF modified HbS by the greatest degree (70%), followed by FUF(24%), and lastly vanillin (15%). FIG. 4 shows the Cation-exchange HPLCstudies of the hemolysate from the 5HMF-reacted SS cells at 5 mMconcentration. The compounds also shifted the OEC of the hemolysate tothe left. These shifts follow the same trend observed during the normalwhole blood studies. 5HMF causes the largest Hb left shift (ΔP₅₀ of −9.4mmHg), followed by FUF (ΔP₅₀=−5.9 mmHg) and lastly vanillin.(ΔP₅₀=−3.1mmHg).

Antisickling Studies of Compounds with Homozygous Sickle Red BloodCells. Upon exposure of SS cell suspensions to only 4% oxygen, in theabsence of test compounds, all cells underwent sickling. In the presenceof vanillin, FUF, and 5HMF (at 5 mM concentrations) the percentage of SScells decreases by 20, 30, and 90%, respectively (Table 4, columns 2 and3). 5HMF inhibited sickling the most, followed by FUF, and vanillin.These results follow the same trend observed in the left shift of theOEC, as well as the compound-HbS adduct formation. Table 4 also showsthe results of the dose-dependent antisickling effect of FUF and 5HMF.Different concentrations of these compounds decreased the formation ofSS cells in a dose-dependent manner. However, unlike 5HMF, FUF did notinhibit cell sickling at lower compound concentrations (1 and 2 mM).TABLE 4 Antisickling Studies with Homozygous Sickle Red BloodCells^(a,b) Inhibition of Sickling of sickling of SS MCV^(c) Compound SScells (%) cells (%) (fl) Control 100 0 61.5 5 mM Vanillin 80 20 ± 6.561.0 1 mM FUF 100 0 60.3 2 mM FUF 100 0 60.0 5 mM FUF 70 30 ± 7.0 62.8 1mM 5HMF 87 13  62.5 2 mM 5HMF 58 42 ± 1.0 61.0 5 mM 5HMF 10 90 ± 5.061.4^(a)The results are the means ± S.E. for 2 measurements.^(b)Antisickling studies with SS cells (10% hematocrit) under 4% oxygen.^(c)MCV is the mean corpuscular volume.

The Effect of Compounds on Homozygous Red Sickle Blood Cell Size—Asshown in Table 4 (column 4) incubation of SS cells with 1, 2, and 5 mMof FUF or 5HMF did not result in changes in cell volumes.

Crystallization Studies. Deoxygenated Hb complexed with either FUF or5HMF crystallized in both T and R2 state conformations. The T stateco-crystallized with FUF and 5HMF show only weak binding of thesecompounds, while the R2 state co-crystallized with these compounds showvery strong binding. With compound to Hb ratios of 4:1, we observed bothT and R2 state crystals in the same crystallization tubes.Interestingly, for 5HMF, more R2 state than T state crystals were alwaysobserved; for FUF the opposite was generally true. However, if a largeexcess of compound is used (≧10 molar excess), nearly all of theco-crystallization experiments with FUF and 5HMF result in only R2 statecrystals. These results suggest that 5HMF is allosterically more potentthan FUF, which is consistent with the biological results.

R2 state native crystals have previously been crystallized under lowsalt conditions (Silva, et al., 1992), but not in high salt conditions.The ensuing structures of the R2 state co-crystals, as already pointedout, have water or O₂ molecules (FUF- and 5HMF-Hb complexes,respectively) coordinated to the Fe atoms. In reality, it ishypothesized that the ligands are actually a mixture of O₂, CO, andwater, as analysis of the R2 state complexes showed the presence ofCOHb, metHb, and oxyHb (60-85%), versus approximately 16% for the Tstate co-crystals. The presence of the ligands in the R2 stateco-crystals could be due to the fact that the anaerobic chamber used inthese experiments may not have been completely devoid of oxygen duringthe crystallization setup. Interestingly, the T state co-crystals thatoccurred in the same solutions as the R2 state co-crystals did not showany residual density for ligand binding to Fe. These observationsunderscore the high-affinity nature of relaxed state Hb compared totense state Hb. These results also clearly show that the R2 quaternarystructure is physiologically important as previously pointed out(Srinivasan & Rose, 1994).

Unlike the T and R2 crystallization results, and quite significantly,repeated R state crystallization experiments did not result in anyco-crystals, especially at high compound concentrations (≧5 molar excessto the Hb tetramer). However, if low compound concentrations (4 molarexcess to the Hb tetramer) were used, very few R state co-crystals forboth FUF and 5HMF were observed, with most of the complex remaining insolution. We should point out that, we were able to easily obtain Rstate native crystals (control experiment without adding any compound)under these same crystallization conditions.

Descriptions of the R2 State Complex Structures. Both FUF-Hb and 5HMF-Hbcomplex structures contain one α1β1-α2β2 tetramer in the asymmetricunit. The R2 state complexes and R2 state native have essentially thesame Hb quaternary structures (rmsds of ˜0.4 Å). However, comparison ofthe R2 state complex structures with R (PDB code 1AJ9) and T (PDB code2HHB) native Hb structures show very significant quaternary structuraldifferences, with rmsd of ˜1.8 Å and ˜3.3 Å, respectively. As previouslyanalyzed and reported by Silva et al., (1992) for R, T and R2 native Hbstructures, the allosteric transitions between the R2 complex structuresand those of the R and T structures show extensive reorganization of theα1β2, α1β2 and β1β2 interfaces in the three Hb states. The structureswere superimposed using the invariant α1β1 dimer (Ca residues) on theBGH frame as defined by Baldwin & Chothia (1979).

Following are detailed descriptions of the interactions between R2 stateHb, and the two compounds, FUF and 5HMF. Both furanic compounds are wellordered with occupancies of approximately 100%. The compounds bind in asymmetry-related fashion at the α-cleft to the two N-terminal αVal1,with the aldehyde functional group forming a covalent bond with the freenitrogen of the valine. Specific interactions between the compounds andthe protein will be discussed for only the α1Val1 binding site, as theother symmetry-related molecule engages in similar, but oppositeinteractions at the α2Val1 binding site. A covalent interaction betweenthe aldehyde and α1Val1 nitrogen directs the furan ring of the compoundstoward the central water cavity. In the FUF-Hb structure, the boundcompound appears to have two alternate conformations that differ byalmost 180°. The aromatic oxygen engages in a very weak intersubunithydrogen bond with α2Ser138 OG (3.6 Å), which serves to tie the twocc-subunits together. Interestingly, if the compound is rotated to itsalternate conformation, the oxygen faces the water cavity and engages ina weak intrasubunit hydrogen bond with α1Ser131 OG (3.5 Å). There arevery few hydrophobic interactions (<3.8 Å) between the furan ring andthe residues Lys127 and Ala130.

Unlike FUF, there is no evidence of compound rotation of the bound 5HMFmolecule, and it assumes a conformation with the ring oxygen facing thewater cavity. The observed interaction between FUF and αSer138 OG istherefore absent in the 5HMF-Hb complex structure. Similar to FUF, thering oxygen of 5HMF engages in a stronger intrasubunit hydrogen bondwith α1Ser131 OG (3.1 Å), compared to FUF binding. In addition, the5-hydroxymethyl substituent of 5HMF also makes a strong intrasubunithydrogen bonding interaction with α1Thr134 OG (2.6 Å); this interactionis absent in the FUF-Hb complex structure. While FUF ties the twoα-subunits together by making a weak intersubunit hydrogen bond withα2Ser138 OG, the two 5HMF molecules are joined together by a strongnetwork of six water-mediated hydrogen bonds, through the hydroxyl andthe ring oxygen moieties that tie the two α-subunits together. Some ofthese water molecules are conserved in the FUF binding pocket, but theyare mobile and do not engage in hydrogen bonding contact with the FUFmolecules. Like the FUF-Hb structure, there are very few hydrophobicinteractions (<3.8 Å) between 5HMF and Hb. The increased number ofinteractions between 5HMF and protein residues (versus FUF), as well asthe strong water-mediated hydrogen bonds that tie the two α-subunitstogether in the 5HMF complex structure, may partly explain why 5HMF isallosterically more potent than FUF.

Descriptions of the T State Complex Structures. Both FUF-HIb and 5HMF-Hbcomplex structures contain one α1β1-α2β2 tetramer in the asymmetricunit. Unlike the R2 complex structures, the T state complexes do notshow clearly defined compound binding. Some examined co-crystals didshow more compound density than others; however repeated model buildingto improve compound density was not successful enough to allow forreliable compound fitting. The T state complexes and T state native haveessentially the same HIb quaternary structures (rmsds of ˜0.4 Å).Superposition of the αVal1 binding sites also shows very few structuraldifferences. Further, we should point out that in our laboratory we havebeen able to easily isolate T state structures that have covalentlybound compounds, with clearly defined electron density at the N-terminalαVal1 using the same T state crystallization conditions as describedabove (Abraham, et al. 1995; Boyiri, et al., 1995).

Descriptions of the R State Complex Structures: Both FUF-Hb and 5HMF-Hbcomplex structures contain one α1β1 dimer in the asymmetric unit.Surprisingly, the few R state crystals that were obtained during theseexperiments show only sparse density at the N-terminal αVal1 bindingsites. Even though the R complex and native quaternary structures areindistinguishable (rmsds ˜0.4 Å), the C-terminus (residues Trp140 andArg141) display significant positional differences. In the complexstructures, these residues have rotated away at the αLys139, displacingαArg141 by almost 180° from its position in the native structure, whileαTyr140 has oriented away by ˜2 Å. αTyr140 OH now engages in hydrogenbonds with αVal93 O and αPro95 N in the complex structures, versusdiagnostic R state native hydrogen bonds with αVal93 N and O. Thereorientation of the C-terminus has led to a much bigger binding cleftin the R complex structures to allow binding of the compound, albeitweak. This contrasts with the native structure, where the C-terminalresidues are found to be sterically blocking this binding site.

Discussion

These studies have identified furan-based derivatives, which arenaturally occurring in a number of foods, as potential new therapeuticsto treat SCD. Results from these studies clearly indicate that thesecompounds possess the ability to: (1) pass through RBC membranes; (2)react with HbS; and (3) allosterically shift the Hb OEC to thehigh-affinity state, which does not form HbS polymers. Furthermore, wehave found that the change in the oxygen affinity of SS cell suspensionscaused by these compounds depends on the degree of binding to HbS; 5HMFshowed the highest amount of compound-HbS adduct, and as expected, wasthe most potent OEC left shifter. Also, the results clearly suggest thatsubstitution, as well as substitution type at the 5-position of thecentral furan ring is important to biological activities. 5HMF, whichpossesses an alkyl alcohol at the 5-position of the furan ring, is morepotent than either 5MF or 5EF (FIG. 2), both of which possesshydrophobic moieties at this position. FUF, without a substitution isthe least potent. This is consistent with the crystallographic results,which indicate that the hydroxyl moiety of 5HMF is intimately involvedin interactions that stabilize the relaxed state. The Hill coefficientsof the modified Hbs are relatively smaller compared to the unmodifiedHb, suggesting a decrease in cooperativity. This is expected because ofthe apparent weakening of interdimer interactions by the binding of thecompounds to the T state, leading to increased oxygen affinity, reducedcooperativity, and a shift toward the high-affinity Hb.

Antisickling Activities of the Furanic Compounds. Results from thescreening of FUF and 5HMF with SS cells show that these compounds havestrong antisickling properties, stronger than other known antisicklingaldehydes. At 5 mM concentration, 5HMF inhibited cell sicklingapproximately 4 and 2.5 times more than vanillin and FUF, respectively.Remarkably, even at 2 mM concentration, 5HMF reduces cell sickling by42%, twice as much as vanillin at 5 mM concentration. 5HMF, whichmodifies HbS the most, is the most potent left-shifting compound, aswell as the most potent antisickling agent; the converse is true forvanillin. Thus, the antisickling action of these compounds seems toresult from their ability to bind to HbS and left shift the OEC towardthe high-affinity state. And even though the antisickling activities of5MF and 5EF (FIG. 2) have not been determined, based onstructure-activity relationships, we predict that both compounds willexhibit antisickling activities that lie between those of FUF and 5HMF.

Also significant is the fact that the compounds did not dehydrate SScells. Polymerization of HbS and the sickling of SS cells are linked tothe intracellular concentration of HbS, therefore, any agent that causesdehydration of RBCs would increase the molar concentration of HbS, andpresumably increase polymer formation. Furthermore, the compounds didnot promote formation of metHb or membrane-associated denatured Hb.

Mechanism for the Antisickling Activities of the Furanic Compounds. Theresults from these studies clearly show that the furanic compoundscovalently bind to and destabilize the T state and/or stabilize therelaxed state HbS. As a result, the allosteric equilibrium left shiftstoward the more soluble, high-affinity HbS in the form of R2conformation. To our knowledge, this is the first such reportedobservation in the literature that shows a compound inducedconformational change of a T state Hb to R2 state Hb, leading toisolation of R2 co-crystals from deoxy Hb solution. We hypothesized thisto be the underlying cause for the observed antisickling activities. Tounderstand the atomic-level mechanism driving the observed biologicalactivities of the compounds, we refer to two landmark publications byAbraham's group (Abraham, et al., 1995; Boyiri, et al., 1995) in whichthe authors hypothesized that agents such as FSA (FIG. 2), that formSchiff base adducts with the N-terminal αVal1 nitrogen of the T state,and provide groups for both salt bridge and hydrogen bonding with theopposite dimer (across the two fold axis), add more constraints to the Tstate. These added constraints shift the allosteric equilibrium towardthe low-affinity T state. In contrast, agents such as DMHB (FIG. 2)which bind to the T state in a similar fashion, but do not engage in anysalt bridges/hydrogen bonding interactions with the opposite dimer, leftshift the OEC. It is hypothesized that these agents disrupt awater-mediated linkage between the N-terminal αVal1 and the C-terminalαArg141 of the opposite dimer, which leads to the destabilization of theT state, and as a result the allosteric equilibrium is shifted towardthe high-affinity R state. Unlike FSA, the furanic compounds lack acarboxylate substituent that would engage in intersubunit stabilizinginteractions when bound to the T state. What is not clear is how thesecompounds bind to the T state, as the crystallographic studies show onlyweak and undefined compound density. However, If we assume that thecompounds bind with the same orientation as observed in the R2 statecomplex structures, a hypothetical fit of 5HMF into the T stateN-terminal αVal1 binding site (with the aid of the weak compounddensity) shows this compound engaging in only intrasubunit interactionswith αThr134 OG1 and CeSer131 OG. Thus it seems the furanic compoundsbind to the N-terminal αVal1 site of the T state, disrupt the nativewater mediated hydrogen bond between αVal1 and αArg141, and destabilizethe T-state. The result of this destabilization is an allosteric shiftto the high-affinity, relaxed Hb in the form of the R2 state. Thismechanism is consistent with the fact that: (1) R2 state crystals wereformed from deoxy Hb/furanic compound solution and (2) T statecrystallization in the absence of furanic compound did not result in R2state crystals. In fact, all of the T state crystals that were isolatedin conjunction with the R2 state crystals had only weak compounddensity—clearly these T state crystals did not have enough boundcompound to effect the allosteric shift. Consistent with thisobservation is the fact that the addition of a large excess of furaniccompound resulted in 100% formation of R2 state co-crystals from thedeoxy Hb complex solution—due to saturation of the binding site of allthe deoxy Hb molecules.

The observed functional and crystallographic results, as well as theproposed mechanism, raise an interesting issue of why R2 state crystalsand not R state crystals form during the T state crystallizationexperiment. Visual analyses and molecular docking studies of theN-terminal αVal1 binding pockets of known T, R and R2 native Hbstructures may shed light on the above question. The native structureswere superimposed using the invariant α1β1 dimer (Ca residues) on theBGH frame (Baldwin & Chothia, 1979). In both the T and R2 states, thebinding pockets of Hb are strikingly larger than that of the R state.Docking studies show that 5HMF can easily fit the T and R2 state bindingpockets without steric interference; in contrast, the N-terminal αVal1binding pocket of the R state native Hb is sterically crowded due to thepresence of the C-terminus residues of Tyr140 and Arg141. Thus, for 5HMFto be able to bind to the R state there must be rearrangements of thebinding pocket residues. This is exactly what occurs in the R statecomplex structures, which show larger binding pockets compared to thenative structure. It seems reasonable that the penalty for rearrangementof the binding pocket residues in the R state should considerably slowthe incorporation of the compound into the binding site (compared to theT and R2 states). These observations may partly explain why binding ofthe furanic compounds to deoxy Hb destabilized this protein to the R2state and not to the R state. Also, we can reasonably assume that thesecompounds bind directly and with higher affinity to the R2 statecompared to the R state, and the compound-HbS adducts observed duringthe HPLC analyses of SS cells are mostly due to the incorporation of thecompounds into the R2 state. We should point out that there is noobvious explanation why we didn't observe R2 state crystals from theaerobic crystallization of compound-COHb solution. However, it is quitepossible that the R2 state complex existed in solution but failed tocrystallize out. This is consistent with the fact that almost all of theaerobic crystallization experiments did not result in crystals, and thefew that did, only produced a few R state crystals, with the majority ofthe complexed species remaining in solution.

Based on the results, it is hypothesized that the observed differencesin the biological activities of the examined furanic compounds are dueto their modes of binding to both the T and R2 state. In the R2 statecomplex structures, 5HMF possesses the ability to stabilize the relaxedconformation to a greater degree than FUF (as discussed above). Modelingof the two compounds into the T state also indicates that 5HMF wouldbind more tightly to the T state than FUF. Therefore, in the absence ofintersubunit salt bridge interactions by these compounds in the T state,it is expected that 5HMF would destabilize the T state more than FUF.

Studies by Abraham, et al. with vanillin (1991), Johnson et al. withpyridoxal (1985), and Park et al. with substituted isothiocyanates,(2003) have suggested that the antisickling effects of these compoundsare due to the direct inhibition of T state polymer formation and/orincreased formation of R state molecules. These studies surmised theformation of R state Hb from the ability of the compounds to shift theOEC to the left. Clearly, our studies unequivocally show that it is theR2 state, rather than the R state, which is formed when the OEC isshifted to the high-affinity Hb. Thus, the mode of action of the furaniccompounds seems to be different from these other antisickling compounds.

Conclusions. A HbS homozygote with a blood volume of 4 L and 25%hematocrit has approximately 5 mmol of HbS. For complete modification ofHbS with 5HMF (mwt=126), 10 mmol will be needed, since two moleculesbind to Hb, translating into 1.26 g of compound. Since 30% modificationof HbS would be enough to achieve clinical benefit, in principle, weneed only administer 378 mg of this compound (assuming the drug targetsHbS only). For a compound like 5HMF, which is non-toxic, a large dosagemay be acceptable, as certain foods that are consumed on a daily basis,such as coffee and caramel products possess concentrations of 5HMF thatsometimes exceed 6 g/kg (Janzowski, 2000). In rats, the acute oral LD₅₀of 5HMF is 2.5 g/kg for males and 2.5-5.0 g/kg for females (US EPA,1992). In comparison, vanillin, which is considered non-toxic, has anacute oral LD₅₀ of 1.58 g/kg. The other furanic compounds also occur innature, and with the exception of FUF, there are no reports aboutpossible adverse effects of MF and EF. The antisickling agents, vanillinand 12C79 also bind covalently to Hb, and both have been shown to beclinically non-toxic (Fitzharris, et al., 1985; Orringer, et al., 1988).Remarkably, 5HMF is more than four times as effective compared tovanillin, which is currently under clinical studies for treatment ofSCD. Thus, the furanic compounds may also be viable drug molecules. Theresults from this study also present a coherent picture of theantisickling potencies and atomic-level mechanisms of new antisicklingagents. With this information, it will be possible to performstructure-activity studies that will result in the development ofanalogs with enhanced potency.

Example 2 In Vivo Antisickling Effect of 5-HMF

In vivo antisickling effect of 5-HMF was investigated using transgenic(Tg) mice that produce human Hb S. Since the blood of wild type mice hasan extremely right-shifted OEC (P50 of mouse blood: 40-44 mm Hg) ascompared with the OEC of human blood (P50 of AA cells: 26.5 mm Hg; SScells: 32 mm Hg), Tg sickle mice that produce approximately equalamounts of human and mouse β-globin and 100% human β^(s)-globin wereused in this study. The P50 of these mice are between 26 and 34 mm Hgdepending on the percentage of mouse β-globin. We used 5% oxygen (5% O₂,95% N₂), because they develop pulmonary sequestration almost exclusivelyupon exposure of these mice to 5% O₂. Although similar changes occursunder oxygen pressures between 6 and 10 mm Hg, not all mice develophypoxia-induced pulmonary sequestration indicating that high numbers ofTg sickle mice are necessary to obtain statistically significantresults. Upon exposure to hypoxia, we determine the percentage ofsickled cells in the blood as well as the survival time. 5HMF (100 mg/kgbody weight) dissolved in a small volume of DMSO was diluted with salinebefore i.p. injection. In the hypoxia experiments, the Tg sickle micewere exposed to hypoxia for up to 1 hr; any surviving mice at 1 hr wereeuthanized by cervical dislocation under anesthesia. In all cases, afterthe mouse died, it was immediately dissected. The heart, lungs, brain,liver, spleen, and kidneys were fixed in 10% phosphate-bufferedformalin. Tissue samples were embedded in paraffin according to standardmethods. Sections were cut and stained by a hematoxylin-eosin solutionfor light microscopy.

Results

FIG. 5 shows the Kaplan-Meir survival plot for control and Tg sicklemice pretreated with 5HMF. Without treatment, Tg sickle mice exposed to5% oxygen die within 15 min due to pulmonary sequestration. Uponpretreatment of Tg sickle mice with 5HMF, more than half of the micesurvived for longer than 25 min. As shown in FIG. 6, the mean survivaltime of control mice was 9.6 3.7 (N=13), while the mean survival time ofmice pretreated with 5HMF was 38.4 mm Hg (n=8). Morphology of SS cellsin the arterial blood at times 0, 10, 20, 30, 40, 55 and 60 min wereinvestigated. The time course of the percentage of sickled cells in thetail artery of control and 5HMF-treated mice is shown in FIG. 7. Changesin the percentage of sickled cells in the arterial blood of one of theTg sickle mice that were exposed to hypoxia (5% oxygen) wereinvestigated. The percentage of an untreated mouse increased from almostzero percentage to over 30% and the animal died in 15 min.Histopathological studies showed that capillaries and small bloodvessels in the lungs of these mice were packed by sickled cells. Thepercentage of sickled cells in one of the mice treated with 5HMF (100mg/kg body weight) showed that although the percentage of sickled cellsincreased, the sickled cells are so called partially oxygenated sickledcells (POSCs) with blunt edges (Asakura, 1994). These cells are flexibleand can pass through capillaries. Upon conversion of POSCs to partiallydeoxygenated sickled cells (PDSCs), they are rigid and trapped in theencountered capillaries. 5HMF not only reduced the formation of POSCs,but also prevented the conversion of flexible POSCs to rigid PDSCs.

Conclusion

Thus in vivo experiments using transgenic sickle mice that produce humansickle Hb showed that pretreatment of the mice with 5HMF(intraperitoneal administration) significantly prolonged the survivaltime under severe hypoxic conditions (5% oxygen). These results indicatethat 5HMF is a new antisickling agent that can passes through red bloodcell membrane, forms Hb adduct and inhibit hypoxia-induced sickling ofSS cells.

Example 3 Generic Synthetic Schemes for Making Representative Compoundsof the Invention

(ref. Vogel's Textbook of Practical Organic Chemistry, 5^(th) edition,1978, by Brian S. Furniss et al.)

The preparation of the monosubstituted aldehydes 5 involved the use ofthe classical Vilsmeier reaction (dimethylformamide/phosphorousdxychloride) with the appropriate substituted starting material.

The preparation of the disubstituted aldehydes, 6 can be accomplished intwo ways. Starting with the appropriate monosubstituded compound 4, willbe lithiated at 78° C. with butyl lithium and quenched with theappropriate bromoalkyl followed by the Vilsmeier reaction to yield therequired aldehyde 6. Alternatively, starting with the protected aldehyde2, compound 6 will be prepared by lithiation with the appropriatebromoalkyl compounds at 78° C. followed by acidic hydrolysis to yieldthe required aldehyde. 6.

Example 4 Prodrug Forms of 5-membered Heterocyclic Antisickling Agentsto Treat Sickle Cell Disease

A. A generic synthetic scheme for making representative prodrugcompounds in Formulas 6 and 11 is given in Scheme 2 below.

General Preparation of 5-membered Heterocyclic Thiazolidine, 8(Huang, T-C; Huang, L-Z; Ho, C-T, J. (1988), Agric. Food Chem. 46, 224)

To a solution of appropriate substituted L-cysteine ethyl esterhydrochloride or cysteamine and ethyl-diisopropyl amine in anhydrousethanol at room temperature will be added5-hydroxymethyl-furan-2-carbaldehyde in anhydrous ethanol. The reactionmixture will be stirred at the same temperature over night. The mixturewill be diluted with water and the product extracted with ethyl acetate.The organic phase will be dried, evaporated, and the product purified byflash chromatography on silica gel to yield the derivative of thethiazolidine compound 8. Where applicable in the case of L-cysteineester analog, the ester substitutent will be hydrolyzed to thecorresponding acid derivative by alkaline hydrolysis using sodiumhydroxide.

General Preparation of 5-membered Heterocyclic Dioxolane, 9

(Abraham, D. J., Safo, M. K., Boyiri, T., Danso Danquah, R., Kister, J.,and Poyart, C. (1995), Biochemistry 34, 15006-15020)

A solution of the appropriate 5-membered heterocyclic aldehyde 7, alkylglycol and catalytic amount of p-toluenesulfonic acid monohydrate willbe stirred at reflux temperature with Dean Stark apparatus for about 12hours. The reaction mixture will be cooled, washed with aqueous sodiumbicarbonate, dried and the solvent evaporated. The product will bepurified by flash chromatography on silica gel to yield the derivativeof the dioxolane, 9.

B. Synthesis of 5-hydroxymethyl-2-furfural-thiazolidine4-carboxylic acidethyl ester (MSDD1), a Prodrug of 5-HMF

A prodrug form of 5HMF,5-hydroxymethyl-2-furfural-thiazolidine-4-carboxylic acid ethyl ester(MSDD1) was synthesized. MSDD1 has the active aldehyde moiety protectedfrom being easily metabolized in the intestines into the inactive acidderivative. This protection leads to increase bioavailability andhalf-life of 5HMF in vivo. For the synthesis, a stirring solution of5HMF (1.51 g, 12 mmol) in absolute ethanol (30 mL) was added a solutionof L-cysteine ethyl ester hydrochloride (2.23 g, 12 mmol) andN-ethyldiisopropylamine (2.55 g, 12 mmol) in absolute ethanol (30 mL)The reaction mixture stirred at room temperature overnight. The mixturewas diluted with water (100 mL) and the product extracted with ethylacetate (3×50 mL). The organic phase was dried, evaporated and theproduct was purified by flash chromatography on silica gel to give 2.77g of product.

C. Oxygen Equilibrium Curve Studies of MSDD1 in Normal Whole Blood:

MSDD1 was tested in normal adult whole blood under in vitro conditionsto find out whether 5HMF with its active aldehyde protected by theL-cysteine ethyl ester would have an effect on the OEC of whole bloodusing multi-point tonometry. The test was conducted as described for5HMF under Example 1, subheading “Oxygen Equilibrium Studies with NormalWhole Blood”. The whole blood oxygen equilibrium studies demonstratethat while 5HMF (with its free active aldehyde unprotected) is able toshift the OEC to the left, the prodrug of 5HMF, (with the aldehydeprotected) clearly does not have effect on the OEC. This suggests thatthe aldehyde, which is the active functional group, is still protectedby the thiazolidine-4-ester group and did not hydrolyze during the invitro test. This is expected since the conditions at which the in vitrostudies were conducted were not expected to lead to the hydrolysis ofthe thiazolidine-4-ester group to free the active 5HMF compound.

Example 5 Methods for Increasing Tissue Hypoxia for Treatment of Cancer

The compounds or the present invention are also useful in the treatmentof cancer. The 5-membered heterocylic aldehydic compounds and theirprotected aldehydic derivatives bind to and destabilize the tense (T)state hemoglobin, resulting in the switch of the allosteric equilibriumin favor of the high-affinity Hb in the form of R2-state Hb. Binding toHb, shifts the oxygen equilibrium curve to the high-affinity R2-statehemoglobin. The compounds thus induce normal tissue and tumor hypoxia bybinding to hemoglobin, increasing its affinity for oxygen and therebyreducing oxygen availability to tissues. Therefore these compounds areof interest as possible potentiators of bioreductive agents and/orhyperthernia in cancer treatment.

The reduction of oxygen available to tissues also leads to protectionagainst radiation damage during X-ray radiation therapy.

The invention includes 5-membered heterocyclic aldehydic and protectedderivatives, that are more potent than 12C79 in stabilizing thehigh-affinity Hb. Additionally, the basis of the allosterism of thesecompounds is understood on molecular level, making it easier to designmore potent effectors. Thus, these compounds improve on known aldehydichypoxic agents by their potency and efficacy.

Example 6 In Vitro Oxygen Equilibrium Studies of Thiophene Analogs ofthe 5-Membered Heterocyclic Anti-sickling Agents with Normal Whole Blood

The following compounds: 5-Bromo-2-thiophenecarboxyaldehyde,4-Bromo-2-thiophenecarboxyaldehyde and3-Methyl-2-thiophenecarboxyaldehyde were purchased from Aldrich ChemicalCompany. Normal blood samples (hematocrit 40%) in the presence of 5 mM5-Bromo-2-thiophenecarboxyaldehyde, 4-Bromo-2-thiophenecarboxyaldehydeand 3-Methyl-2-thiophenecarboxyaldehyde (solubilized in DMSO) wereequilibrated at 37° C. for 1 hr. The samples were then incubated in IL237 tonometers (Instrumentation Laboratories, Inc. Lexington, Mass.) forapproximately 10 min at 37° C., and allowed to equilibrate at oxygentensions 7, 20, and 60 mmHg. The samples were aspirated into an IL 1420Automated Blood Gas Analyzer and an IL 482 or IL 682 Co-oximeter(Instrumentation Laboratories) to determine the pH, pCO₂, pO₂ and the Hboxygen saturation values (sO₂). The pO₂ and sO₂ values at each oxygensaturation level were then subjected to a non-linear regression analysisusing the program Scientist (Micromath, Salt Lake City, Utah) tocalculate the P₅₀ and Hill coefficient values (n₅₀). P₅₀ is the oxygenpressure in mmHg at which Hb is 50% saturated with oxygen.

Results: As shown in Table 5, all three thiophene compounds shift theOEC curve to the left, similar to the above studied furanic compounds.The studies also indicate that the thiophene compounds (like the furaniccompounds) possess the ability to: (1) pass through RBC membranes; (2)react with HbS; and (3) allosterically shift the Hb OEC to thehigh-affinity state, which does not form HbS polymers. Also, the resultssuggest that substitution, as well as substitution type on the centralthiophene ring is important to biological activities. This studies showthat the thiophene analogs are also potential anti-sickling agents.TABLE 5 Results of in Vitro Whole Blood Studies with Thiophene AldehydicCompounds Compound P₅₀c P₅₀d ΔP₅₀ n₅₀

27.73 21.96 20.24  −6.63 2.35

27.73 16.85 17.49 −10.56 2.41

28.10 21.55 20.63  −7.01 2.35The analyses were carried out at a final compound concentration of 5 mM.P₅₀c control value in the absence of compound in mmHg. P₅₀d value in thepresence of compound in mmHg. ΔP₅₀ = P₅₀d − P₅₀c) in mmHg. The Hillcoefficient at 50% saturation (n₅₀) in the presence of compound. Eachmeasurement was repeated at least twice.

References for Examples 1-6

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Example 7 General Preparation of 4-Alkyloxymethyl or Alkylnoic AcidDerivatives of Furan-2-Carbaldehyde

The key precursor for 4, (4-alkyloxymethyl-furan-2-yl)-methanol, 3, isprepared by starting with 4-hydroxymethyl-furan-2-carbaldehyde, 1.O-bromination with the appropriate alkyl bromide in DMF at 60-80° C.yields 2. Reduction of the aldehydic moiety with sodium borohydridewould give the main precursors 3. Vilsmeier reaction of 3 would affordthe product, 4. In the case of compound 4 with acidic side chains(alkylnoic acid derivatives), bromo alkyl ester would be employedinstead of alkyl bromide and the final products obtained after basichydrolysis of the ester side chain with sodium hydroxide. Other analogswould be prepared in a similar manner as described.

General Preparation of Pyridyl Derivatives of Furan-2-Carbaldehydes.

Appropriately substituted methylchloropyridine is condensed with4-hydroxymethyl-furan-2-carbaldehyde in DMF at 70-80° C. in the presenceof potassium carbonate to give the intermediate compound 2. Reduction ofthe aldehydic moiety with sodium borohydride would give the mainprecursor 3. Vilsmeier reaction of 3 would afford the main product,pyridyl substituted furan-2-carbaldehyde 4. Other analogs would beprepared in a similar manner as described.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1. A compound of formula

where R1 is CHO or an aldehyde protecting group; R2 and R3 are the sameor different and are H, OH, alkyl, alkoxy, hydroxy-alkyl, halogen, aryl,or O-aryl; R4 and R5 are the same or different and are a substituted orunsubstituted aromatic or heteroaromatic moiety, a substituted orunsubstituted alkyl or heteroalkyl ring moiety, a substituted orunsubstituted alkyl or alkylnoic acid or ester moiety; m=1-6; X=NH, O,S, Se, or P; and wherein Y=a chemical bridge which includes one to fourchemical moieties selected from the group consisting of CH2, CO, O, S,NH, NHCO and NHCONH; Z=(CH)n where n=1-4; and positions of Y and Z areinterchangeable.
 2. The compound of claim 1, wherein said compound isselected from the group consisting of


3. The compound of claim 1, wherein R1 is a heterocyclic ring aldehydeprotecting group, and the compound is of the formula


4. The compound of claim 3, wherein said compound is selected from thegroup consisting of


5. The compound of claim 1, wherein R1 is a heterocyclic ring aldehydeprotecting group, and the compound is of the formula

wherein R6 and R7=H or alkyl or ester and may be the same or different.6. The compound of claim 5, wherein said compound is selected from thegroup consisting of


7. A method for treating sickle cell disease in a patient in needthereof, comprising the step of administering to said patient a compoundof formula

where R1 is CHO or an aldehyde protecting group; R2 and R3 are the sameor different and are H, OH, alkyl, alkoxy, hydroxy-alkyl, halogen, aryl,or O-aryl; R4 and R5 are the same or different and are a substituted orunsubstituted aromatic or heteroaromatic moiety, a substituted orunsubstituted alkyl or heteroalkyl ring moiety, a substituted orunsubstituted alkyl or alkylnoic acid or ester moiety; m=1-6; X=NH, O,S, Se, or P; and wherein Y=a chemical bridge which includes one to fourchemical moieties selected from the group consisting of CH2, CO, O, S,NH, NHCO and NHCONH; Z=(CH)n where n=1-4; and positions of Y and Z areinterchangeable; and wherein said compound is administered in sufficientquantity to ameliorate symptoms of sickle cell disease.
 8. The method ofclaim 7, wherein said compound is selected from the group consisting of


9. The method of claim 7 wherein said compound is


10. The method of claim 9, wherein said compound is selected from thegroup consisting of


11. The method of claim 7, wherein said compound is


12. The method of claim 11, wherein said compound is selected from thegroup consisting of


13. A method for inducing hypoxia in tissue, comprising the step ofadministering to said tissue a compound of formula

where R1 is CHO or an aldehyde protecting group; R2 and R3 are the sameor different and are H, OH, alkyl, alkoxy, hydroxy-alkyl, halogen, aryl,or O-aryl; R4 and R5 are the same or different and are a substituted orunsubstituted aromatic or heteroaromatic moiety, a substituted orunsubstituted alkyl or heteroalkyl ring moiety, a substituted orunsubstituted alkyl or alkylnoic acid or ester moiety; m=1-6; X=NH, O,S, Se, or P; and wherein Y=a chemical bridge which includes one to fourchemical moieties selected from the group consisting of CH2, CO, O, S,NH, NHCO and NHCONH; Z=(CH)n where n=1-4; and Y and Z areinterchangeable; wherein said compound is administered in a quantitysufficient to induce hypoxia.
 14. The method of claim 13, wherein saidmethod is used to potentiate the action of bioreductive agents orhyperthermia during cancer treatment.
 15. The method of claim 13,wherein said method is used to protect against radiation damage duringX-ray radiation therapy.